Article Figures & Data
Figures
- Figure 1.
Impact of FAD2 intron suppression on fatty acid composition of mature soybean seeds. For each line, 24 mature seeds were individually analyzed by GC-FAME. The values are averages of fatty acid mass percentage. Bars from left to right: CTRL, MOC-1, MOC-2, MOC-3, MOC-4, and MOC-5.
- Figure 2.
Impact of FAD2 intron suppression during seed development on 18:1 content and FAD2 transcripts. Developing seeds from greenhouse-grown plants were sorted into 10 stages as defined in “Materials and Methods,” from 10 mg fresh weight (stage 0) to maximum weight (290 mg) green seeds (stage 6), from the onset and midpoint of seed desiccation (stages 7 and 8, respectively), to fully mature dry seeds (stage 9). A, Oleic acid composition via GC-FAME. B to E, Total RNA from all stages was isolated and subjected to gene-specific quantitative RT-PCR, with the respective specificities indicated. All data are averages of two lines. Circles represent CTRL plants, and squares represent averaged homozygous suppressed lines MOC-1 and MOC-2.
- Figure 3.
Genomic DNA gel-blot analysis of transgenic lines. Genomic DNA was digested with BamHI, SpeI, and SacI as indicated. After agarose gel electrophoresis and transfer, the genomic DNA was hybridized to the 420-bp FAD2-1A intron probe. Mr markers are indicated to the left. From left to right, MOC-1, MOC-2, MOC-3, MOC-4, MOC-5, and CTRL are indicated by numbers and C, respectively. The signals shared between CTRL and the transgenic lines represent the endogenous soy FAD2 sequence. Each of the transgenic lines also shows one extra band corresponding to the transgene insert.
- Figure 4.
Map for the transgenic locus for MOC-1. The locus consists of two copies of the FAD2-1A intron suppression cassette in an inverted repeat orientation. The left copy is complete, and the right copy has a deletion of the 3′ terminal 220 bp of the H6 3′ UTR as well as the entire right border (RB) sequence. The H6 3′ UTR sequence targeted by Invader assay for copy number estimation is indicated. The black triangles indicate the locations and orientations of the inverted repeat primers. The map is not to scale. LB, Left border.
- Figure 5.
Detection of inverted repeat transcripts in the seeds of MOC-1. Total RNA was isolated from developing and mature seeds, treated with DNase, and used as template for RT, followed by 20 cycles of PCR. Products were transferred to a membrane and probed with DNA sequence from the cotton H6 3′ UTR. The nontransgenic CTRL line shows no signal, and a signal is present in MOC-1. The signal was absent when the RT step for MOC-1 was omitted (−RT), demonstrating the absence of genomic DNA contamination.
- Figure 6.
RNA gel-blot analysis of FAD2 transcripts in developing and mature soy seeds. A and B, Total RNA was isolated from seed stages 3, 6, and 9 and leaves (L), separated by agarose gel electrophoresis, transferred to a membrane, and hybridized to the 420-bp FAD2-1A intron DNA probe (A) or a 316-bp FAD2-1A exon DNA probe (B). Mr markers are indicated at the left. C, The ethidium bromide fluorescence of the stained RNA.
- Figure 7.
Small RNA analysis of developing and mature soy seeds. Total RNA was isolated as described, separated on a 17% acrylamide gel, and transferred to a nylon membrane. Top, the blot was hybridized with the 420-bp FAD2-1A intron RNA probe. The nontransgenic CTRL line shows no signal at any stage. MOC-1 shows the accumulation of FAD2-1A intron siRNAs at each seed stage. In comparison, a conventional RNAi transformant (INT-1), containing the same FAD2-1A intron suppression trigger sequence, also shows accumulation of FAD2-1A intron siRNAs. Middle and bottom, small RNA blot probed with a soy miR159 probe and ethidium bromide-stained ribosomal RNA to show equal loading. nt, Nucleotides.
